Methods of identifying genetic mutations associated with charcot-marie-tooth neuropathy type 1C

ABSTRACT

In one aspect, the invention provides methods of identifying genetic mutations that are associated with peripheral neurological disease. The methods comprise identifying a difference between a nucleic acid sequence of a small integral protein of the lysosome/late endosome (“SIMPLE”) gene from a mammalian subject exhibiting peripheral neuropathy and a nucleic acid sequence of a SIMPLE gene from a subject which is not exhibiting peripheral neuropathy, wherein the difference is a genetic mutation associated with peripheral neurological disease. In another aspect, isolated nucleic acid molecules encoding SIMPLE missense mutations are provided. In another aspect, a method of screening a subject to determine if the subject has a genetic predisposition to develop Charcot-Marie-Tooth type 1C neuropathy is provided. In another aspect, the invention provides kits for determining susceptibility or presence of Charcot-Marie-Tooth type 1C neuropathy in a mammalian subject.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of co-pending application Ser. No.10/756,194, filed Jan. 13, 2004, which claims the benefit of ProvisionalApplication No. 60/440,399, filed Jan. 13, 2003. The benefit of thepriority of the filing dates of each application is hereby claimed under35 U.S.C. §§120 and 119, respectively. Each application is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Grant No. NS38181awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

The present invention relates to methods and kits for identifyingsubjects susceptible to Charcot-Marie-Tooth Neuropathy.Charcot-Marie-Tooth (CMT) neuropathy, also called Hereditary Motor andSensory Neuropathy (HMSN), is a clinically and genetically heterogeneousgroup of inherited peripheral neuropathies leading to progressive distalmuscle weakness and sensory loss. CMT is frequently transmitted in anautosomal dominant manner. An estimated 1 in 2,500 persons has a form ofCMT, making it a major diagnostic category within neurogenetic diseases(She, H., Clin. Genet. 6:98-118 (1974)). A current classification systemdivides CMT neuropathy into CMT type 1 (CMT1), which is characterized bydemyelination and reduced nerve conduction velocities (NCVs)(typically<40 meters/sec), and CMT type 2 (CMT2), which denotes patients withaxonal neuropathy, lack of myelin abnormalities in pathologic specimens,and nearly normal nerve conduction velocities (Dyck and Lambert, Arch.Neurol. 18:603-618 (1968)). Onset of symptoms associated with CMTtypically occurs in adolescence or early adulthood; however,presentation may be delayed until mid-adulthood. The severity ofsymptoms is variable, even among members of the same family, withgradual progression of symptoms. Typical CMT symptoms include pes cavus,distal muscle weakness and atrophy, absent or diminished deep tendonreflexes, and mild sensory loss.

CMT1 has been divided into five subtypes (CMT1A-D, X), based on geneticlinkage analysis; however, the CMT1 subtypes are clinicallyindistinguishable. CMT1A is associated with a 1.4 megabase (Mb)duplication on chromosome 17p11.2-p12 and a gene dosage effect forperipheral myelin protein (PMP22) (Matsumami et al., Nat. Gen.: 176-179(1992)). CMT1B is associated with mutations in the myelin protein zerogene (MPZ) (Hayasaka et al., Nat. Genet. 5:31-34 (1993). CMT1D isassociated with mutations in the early growth response 2 element gene(EGR2) (Warner et al., Nat. Gen. 18:382-384 (1998) and CMTX isassociated with mutations in the connexin 32 (Cx32) gene (Bergoffen etal., Science 262:2039-2042 (1993). Recently, mutations in theganglioside-induced differentiation-associated protein 1 gene (GDAP1)have been associated with autosomal recessive demyelinating CMT as wellas autosomal recessive axonal CMT with vocal cord paralysis (Nelis, E.,et al., Neurology 59(12):1835-6 (2002). Patients that exhibit symptomsassociated with CMT1, but that lack mutations in these known genes, havebeen assigned to subtype CMT1C.

Given the prevalence of CMT1 cases not linked to any known genetic loci,there is a need to identify genetic mutations associated with the CMT1syndrome that can be used in a genetic screen to identify subjectssusceptible to CMT1 neuropathy. The present inventors have discoveredindividuals with mutations in the small integral membrane protein of thelysosome/late endosome (“SIMPLE”) gene and have established a molecularlinkage for CMT1C.

SUMMARY

In accordance with the foregoing, in one aspect the present inventionprovides methods of identifying genetic mutations that are associatedwith peripheral neurological disease in a mammalian subject, the methodscomprising identifying a difference between a nucleic acid sequence of asmall integral membrane protein of the lysosome/late endosome (“SIMPLE”)gene from a first mammalian subject exhibiting peripheral neuropathy anda nucleic acid sequence of a SIMPLE gene from a second mammalian subjectwhich is not exhibiting peripheral neuropathy, wherein the first andsecond mammalian subjects are members of the same species, and whereinthe difference between the nucleic acid sequences is a genetic mutationthat is associated with peripheral neurological disease. In someembodiments of this aspect of the invention, the method furthercomprises determining whether the identified mutations co-segregate withperipheral neuropathy.

In another aspect, the present invention provides isolated nucleic acidmolecules encoding a SIMPLE protein comprising a missense mutation, theisolated nucleic acid molecules comprising a sequence selected from thegroup consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

In another aspect, the present invention provides isolated SIMPLEpolypeptides comprising a missense mutation, the isolated SIMPLEpolypeptides comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.

In another aspect, the present invention provides a nucleic acid probefor detecting a SIMPLE gene consisting of a nucleic acid sequenceselected from the group consisting of a nucleic acid sequence spanningnucleotide 91 to nucleotide 140 of SEQ ID NO: 4, or the complementthereof, and SEQ ID NO: 6, or the complement thereof.

In another aspect, the present invention provides nucleic acid primermolecules consisting of SEQ ID NO: 18 and SEQ ID NO: 19 which are usefulfor amplifying exon 3 of a SIMPLE gene.

In another aspect, the present invention provides methods of screening amammalian subject to determine if said subject has a geneticpredisposition to develop, or is suffering from Charcot-Marie-Toothneuropathy type 1C (CMT1C). The method of this aspect of the inventioncomprises analyzing the nucleic acid sequence of a SIMPLE gene in amammalian subject to determine whether a genetic mutation that isassociated with CMT1C is present in the nucleic acid sequence, whereinthe presence of an identified genetic mutation in the SIMPLE gene thatco-segregates with CMT1C indicates that the mammalian subject has agenetic predisposition to develop CMT1C or is suffering from CMT1C.

In another aspect, the invention provides a kit for determiningsusceptibility or presence of CMT1C in a mammalian subject based on thedetection of a mutation in a SIMPLE gene, said kit comprising (i) one ormore nucleic acid primer molecules for amplification of a portion of aSIMPLE gene and (ii) written indicia indicating a correlation betweenthe presence of said mutation and risk of developing CMT1C. In someembodiments, the kit detects the presence or absence of a mutation inthe SIMPLE gene selected from the group consisting of G112S, T115N andW116G.

The invention thus provides methods, reagents and kits for identifyinggenetic mutations in a SIMPLE gene and thereby facilitates diagnosis ofCharcot-Marie-Tooth neuropathy and identification of carriers of thegenetic defect. The nucleic acid molecules of the invention are usefulas probes to identify genetic mutations in the SIMPLE gene and havetherapeutic utility for identifying compounds that can be used to treatCharcot-Marie-Tooth neuropathy.

DETAILED DESCRIPTION

Unless specifically defined herein, all terms used herein have the samemeaning as they would to one skilled in the art of the presentinvention. Practitioners are particularly directed to Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Press, Plainsview, N.Y., and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York (1999) for definitionsand terms of art.

The following definitions are provided in order to provide clarity withrespect to the terms as they are used in the specification and claims todescribe the present invention.

As used herein, the term “peripheral neuropathy” refers to peripheralnerve damage, including sensory and motor nerve damage, producing avariety of symptoms including for example, muscle weakness, numbness,paresthesia and pain in the arms, hands, legs and/or feet, pes cavus andreduced nerve conduction velocity.

As used herein, the term “peripheral neurological disease” refers to theclinical manifestation of a typically slowly progressive peripheralneuropathy.

As used herein, the term “paresthesia” refers to abnormal sensationssuch as burning, tickling, pricking or tingling.

As used herein, the term “pes cavus” refers to a deformity of the footproducing a high arch that does not flatten with weightbearing. Thedeformity can be located in the forefoot, midfoot, or hindfoot or in acombination of these sites.

As used herein, the term “proband” refers to the family member throughwhom a family's medical history comes to light.

As used herein, the term “Charcot-Marie-Tooth neuropathy” or (“CMT”)refers to a peripheral (motor and sensory) neuropathy without anestablished acquired (non-genetic) cause as described in Sherer et al.,(2003) The Molecular and Genetic Basis of Neurological Diseases, 3d Ed.,Oxford Butterworth Hiemen Press, pp. 435-453. Unexplained chronicprogressive neuropathy in individuals with a negative family history forperipheral neuropathy may represent an instance of inherited CMT on thebasis of a new dominant mutation, or a single occurrence of an autosomalrecessive or X-linked disorder in a family. CMT patients typicallyexperience slowly progressive symptoms ranging from muscle weakness inthe arms, legs, hands and feet, decreased muscle bulk, reduced tendonreflexes and sensory loss. Individuals with CMT may also have footdeformities, such as pes cavus, high arches, hammertoes, inverted heel,flat feet and other orthopedic problems such as mild scoliosis or hipdysplasia.

As used herein, the term “Charcot-Marie-Tooth Neuropathy type 1” or(“CMT1”) includes a large group of inherited autosomal dominantdisorders characterized by peripheral nerve demyelination affectingperipheral (motor and/or sensory) nerves. Hallmarks of CMT1 includereduced nerve conduction velocities (typically less than 40 meters/sec),and nerve biopsies that display “onion bulb” formation. Studies havedetermined that reduction in nerve conduction velocity can be accuratelydetermined in most cases by the age of five years (Nicholson, G. A.Neurology 41:547-552 (1991).

As used herein, the term “Charcot-Marie Tooth Neuropathy type 1C” or(“CMT1C”) refers to one of five clinically indistinguishable subtypes ofCharcot-Marie-Tooth Neuropathy type 1 that is designated as type C basedon the lack of molecular linkage to genetic loci associated withsubtypes CMT1A, CMT1B, CMT1D, and CMT1X and/or based on the presence ofmutation(s) in the SIMPLE gene.

As used herein, the term small integral membrane protein of thelysosome/late endosome “SIMPLE,” also known as thelipopolysaccharide-induced TNF-alpha factor “LITAF,” or p53-induced gene“PIG7” refers to any gene that encodes the SIMPLE/LITAF/PIG7 protein.Some SIMPLE genes useful in the practice of this invention are at least90% identical to the nucleic acid sequence set forth in SEQ ID NO: 3.Some SIMPLE genes useful in the practice of this invention are at least95%, or at least 99% identical to the nucleic acid sequence set forth inSEQ ID NO: 3.

As used herein, the term “primer” means a polynucleotide which can serveto initiate a nucleic acid chain extension reaction. Typically, primershave a length of 5 to about 50 nucleotides, although primers can belonger than 50 nucleotides.

As used herein, the term “sequence identity” or “percent identical” asapplied to nucleic acid molecules is the percentage of nucleic acidresidues in a candidate nucleic acid molecule sequence that areidentical with a subject nucleic acid molecule sequence (such as thenucleic acid molecule sequence set forth in SEQ ID NO: 3), afteraligning the sequences to achieve the maximum percent identity, and notconsidering any nucleic acid residue substitutions as part of thesequence identity. No gaps are introduced into the candidate nucleicacid sequence in order to achieve the best alignment. Nucleic acidsequence identity can be determined in the following manner. The subjectpolynucleotide molecule sequence is used to search a nucleic acidsequence database, such as the Genbank database, using the programBLASTN version 2.1 (based on Altschul et al., Nucleic Acids Research25:3389-3402 (1997)). The program is used in the ungapped mode. Defaultfiltering is used to remove sequence homologies due to regions of lowcomplexity as defined in Wootton, J. C., and S. Federhen, Methods inEnzymology 266:554-571 (1996). The default parameters of BLASTN areutilized.

As used herein, the term “genetic mutation” is an alteration of thewild-type SIMPLE gene sequence deposited in GenBank, provided as SEQ IDNO: 3 that is not a recognized polymorphism. A polypmorphism typicallyhas a population frequency of greater than 1% in mammalian controlsubjects of the same species that do not exhibit peripheral neuropathy.

In one aspect, the present invention provides methods of identifyinggenetic mutations that are associated with peripheral neurologicaldisease in a mammalian subject. The methods of this aspect of theinvention comprise the step of identifying a difference between anucleic acid sequence of a small integral membrane protein of thelysosome/late endosome (“SIMPLE”) gene from a first mammalian subjectexhibiting peripheral neuropathy and a nucleic acid sequence of a SIMPLEgene from a second mammalian subject which is not exhibiting peripheralneuropathy, wherein the first and second mammalian subjects are membersof the same species, and wherein the difference between the nucleic acidsequences is a genetic mutation that is associated with peripheralneurological disease. In some embodiments, the method further comprisesthe step of determining whether the identified genetic mutationco-segregates with peripheral neuropathy.

The methods of this aspect of the invention are useful to identifygenetic mutations associated with hereditary peripheral neurologicaldisease in any mammalian subject, particularly human subjects. Forexample, the methods of the invention may be used to identify geneticmutations in the SIMPLE gene that are associated (i.e., where themutation is found to occur in subjects predisposed to develop hereditaryperipheral neurological disease and the mutation is not found insubjects not predisposed to develop hereditary peripheral neurologicaldisease) with the occurrence of hereditary peripheral neurologicaldisease in individuals at risk for developing this disease.

The present inventors have discovered that mutations in the SIMPLE genelocus are responsible for a portion of cases of the peripheralneurological disease Charcot-Marie-Tooth Neuropathy type 1C (CMT1C).Previously, patients were designated as subtype CMT1C based on theabsence of mutations in genetic loci known to be associated with CMT1,such as the peripheral myelin protein 22 (PMP22) gene (associated withCMT1A), the myelin protein zero gene (MPZ) (associated with CMT1B), theearly growth response 2 element gene (EGR2) (associated with CMT1D) orthe connexin 32 (Cx32) (associated with CMTX). SIMPLE was identified asa candidate gene for CMT1C based in part on chromosomal mapping to a 9cM region on chromosome 16p as described in Example 1.

The SIMPLE gene appears to be almost ubiquitously expressed (Moriwaki etal., J. Biol Chem 276:23065-23076 (2001), however the biologicalfunction of the SIMPLE protein is currently unknown. The protein encodedby the SIMPLE gene possesses a putative membrane association domainflanked by two putative CXXC motifs, known as high affinity zinc bindingmotifs (Collet et al., J. Biol. Chem 2:2, (2003). In addition, theN-terminus of the SIMPLE protein contains two PPXY motifs (WW domainbinding motif) that have been shown to interact with Nedd4, an E3ubiquitin ligase that plays a role in ubiquitinating membrane proteins(Jolliffe et al., Biochem J 351:557-565 (2000)). The SIMPLE protein isidentical to a protein previously described aslipopolysaccharide-induced TNF-alpha factor (“LITAF”). Moriwaki et al.,J. Biol Chem 276:23065-23076 (2001). LITAF was originally cloned as aputative nuclear transcription factor involved in binding to a criticalregion of the TNF-alpha promoter (Myokai et al., Proc Natl Acad Sci96:4518-4523 (1999)).

The human SIMPLE gene consists of four exons spanning a genomic intervalof 37.75 kilobases, with a start codon located in exon 2. The SIMPLEcDNA coding sequence is provided herein as SEQ ID NO: 1 whichcorresponds to nucleotides 234-719 of GenBank accession number AB034747.Disclosed herein are nucleic acid mutations numbered sequentially withrespect to the first nucleotide of SEQ ID NO: 1. The SIMPLE proteinencoded by SEQ ID NO: 1 is provided herein as SEQ ID NO: 2. Disclosedherein are amino acid mutations numbered sequentially with respect tothe first amino acid residue of SEQ ID NO: 2. The entire 37.75 kilobasegenomic locus that encompasses the SIMPLE gene is provided herein as SEQID NO: 3. With respect to the first nucleotide in SEQ ID NO: 3, the fourexons are as follows: exon 1: nucleotides 1 to 228; exon 2: nucleotides29,639 to 29,863; exon 3: nucleotides 32,685 to 32,840; and exon 4:nucleotides 36,629 to 37,775. The start codon is in exon 2 at nucleotide29,644. The nucleic acid sequence encoding exon 3 (nucleotides 29,639 to29,863 of SEQ ID NO: 3) is provided herein as SEQ ID NO: 4, whichencodes the amino acid sequence provided as SEQ ID NO: 5.

The present inventors have identified several missense mutations (forexample, G112S, T115N and W116G) in SIMPLE that cause a portion of CMT1Ccases. The patients with these mutations in SIMPLE (as further describedin Examples 1 and 2) exhibited peripheral neuropathy and met widelyaccepted criteria for CMT1 including distal muscle weakness and atrophy,depressed deep tendon reflexes and sensory impairment (see Dyck, P. J.,and E. H. Lambert, Arch. Neurol. 18:619-625 (1968)). The three missensemutations G112S, T115N, and W116G are clustered in a conserved region ofexon 3 encompassing seven amino acids as shown in Table 1. The aminoacid sequence of the wild-type human SIMPLE protein AGALTWL (SEQ ID NO:7) is identically conserved in human, mouse, rat and chicken (Street etal., Neurology 60:22-27). As shown in Table 1, the tight clustering ofmutations within exon 3 of the SIMPLE gene suggest this domain, which isimmediately adjacent to a membrane association domain, is critical toperipheral nerve function. The first column of Table 1 provides the CMT1pedigree from which each mutant was identified. The second columnprovides the altered (underlined) amino acid residue in each CMT1pedigree.

TABLE 1 CONSERVED AMINO ACID REGION OF THE SIMPLE PROTEIN CONTAININGMISSENSE MUTATIONS CMT1 Pedigree/ Amino Acid Mutation Nucleic AcidSequence Sequence Wild-type GCCGGTGCTCTGACCTGGCTG AGALTWL human SEQ IDNO: 6 SEQ ID NO: 7 K1551, K1552, GCCAGTGCTCTGACCTGGCTG ASALTWL PN282G112S SEQ ID NO: 8 SEQ ID NO: 9 K1550 GCCGGTGCTCTGAACTGGCTG AGALNWLT115N SEQ ID NO: 10 SEQ ID NO: 11 K2900, K1910 GCCGGTGCTCTGACCGGGCTGAGALTGL W116G SEQ ID NO: 12 SEQ ID NO: 13

The practice of this aspect of the invention is therefore useful toidentify additional mutations in the SIMPLE gene that are associatedwith peripheral neurological diseases such as, for example, CMT (type 1or type 2), Dejerine-Sottas disease, congenital hypomyelinationneuropathy, and hereditary neuropathy with liability to pressurepalsies. By way of illustrative example, Dejerine-Sottas disease (DSD)is a peripheral neurological disease with clinical features that overlapwith those of severe CMT1. Molecular studies indicate that DSD, likeCMT, shows genetic heterogeneity and shares several genetic loci incommon with those implicated for CMT1, including PMP22, MPZ and EGR2, aswell as periaxin (PRX) (Boerkoel et al., Am J Hum Genet 68: 325-33(2001). Therefore, the methods of this aspect of the invention may beused to identify genetic mutations in SIMPLE that are associated withDejerine-Sottas disease or other peripheral neurological diseases. Insome patients with peripheral neuropathy, mutations in SIMPLE may occurin combination with a mutation in another gene known to be associatedwith peripheral neurological disease, such as, for example, PMP22, Cx32,MPZ, EGR2, GDAP, and PRX as described herein. For example, mutations inthe SIMPLE gene may act to increase or decrease the clinical severity ofa peripheral neurological disease that has previously been associatedwith a mutation in a gene other than SIMPLE.

In the practice of this aspect of the method of the invention, anymethod of obtaining reliable nucleic acid sequence data from a mammaliansubject exhibiting peripheral neuropathy may be utilized. For example,reliable sequence data may be obtained from existing databases ofsequence data, or alternatively, a reliable nucleic acid assay that willidentify a genetic mutation in the SIMPLE gene may be utilized.

In one embodiment of the method of the invention, a genetic mutation isdetected by amplification of all or part of the SIMPLE gene from genomicDNA followed by sequencing of the amplified DNA. For example, each ofthe four exons of the SIMPLE gene may be amplified individually or incombination using as template genomic DNA from a test subject exhibitingperipheral neuropathy. A method of amplification which is well known bythose skilled in the art is the polymerase chain reaction (PCR) (seeCurrent Protocols in Molecular Biology, Ausubel, F. M. et al., JohnWiley & Sons; 1995). Alternative amplification techniques may also beused in the method of this aspect of the invention, such as the ligasechain reaction (LCR) (Wu and Wallace, Genomics 4:560-569 (1989)), stranddisplacement amplification (SDA) (Walker et al., Proc. Nat'l. Acad. Sci.USA 89:392-396 (1992)), self-sustained sequence replication (3SR) (Fahyet al., PCR Methods Appl. 1:25-33 (1992)), and branched chainamplification which are known and available to persons skilled in theart.

The PCR process involves the use of pairs of primers, one for eachcomplementary strand of the duplex DNA (wherein the coding strand isreferred to as the “sense strand” and its complementary strand isreferred to as the “anti-sense strand”), that will hybridize at siteslocated on either side of a region of interest in a gene. Chainextension polymerization is then carried out in repetitive cycles toincrease the number of copies of the region of interest exponentially.Primers useful in the practice of the method of the invention comprisepolynucleotides that hybridize to a region of a SIMPLE gene, which canserve to initiate a chain extension reaction. A “primer pair” is a pairof primers which specifically hybridize to sense (coding) and antisense(non-coding) strands of a duplex polynucleotide to permit amplificationof the region lying between the primers of the pair. Primers useful inthe practice of this aspect of the invention comprise a polynucleotideof any size that is capable of hybridizing to SEQ ID NO: 1, SEQ ID NO: 3or SEQ ID NO: 4 under conditions suitable for PCR amplification and/orsequencing. In a preferred embodiment, primers useful in the practice ofthis aspect of the invention range from about 5 to 50 bp or longer ofcontinuous sequence chosen from SEQ ID NO: 1, SEQ ID NO: 3, or SEQ IDNO: 4. Table 2 describes sets of primers useful for PCR amplifying andsequencing of each of the four exons of the SIMPLE gene from genomicDNA. The first column of Table 2 describes the exon to be amplified andthe second column and third columns provide the nucleotide sequence ofthe forward and reverse primers used to amplify the exon and theircorresponding SEQ ID NOs. Tm refers to the melting temperature of theoligonucleotide pair. The expected PCR product size in base pairs (bp)for each PCR amplification is provided in the fifth column. Example 1provides a non-limiting example of this embodiment of the method of theinvention.

TABLE 2 PRIMERS FOR EXON FRAGMENT AMPLIFICATION AND SEQUENCING OF THESIMPLE GENE Size Exon Forward Primer Reverse Primer Tm bp 1 1F 5′TCAGAAACAAAACCA- 1R 5′ GTCCCACCAGCACCTACCC 3′ 59.7 337 AAACAAACA 3′ (SEQID NO: 14) (SEQ ID NO: 15) 2 2F 5′ CAACTGAATTTCTTATCT- 2R 5′GTAAAACTGGAACGTACTGG 3′ 55 387 GG 3′ (SEQ ID NO: 16) (SEQ ID NO: 17) 33F 5′ ATAGCCAGACGATGA- 3R 5′ ATGGTGCAGTTGAGAACC 3′ 53 385 ACG 3′ (SEQ IDNO: 18) (SEQ ID NO: 19) 4 4F 5′ GAACATTTTGGCAGC 3′ 4R 5′TAATGGTAGGCACTAAAGG 3′ 59 636 (SEQ ID NO: 20) (SEQ ID NO: 21)

In one embodiment of the method of the invention, after amplification,genetic mutations are detected in the amplified DNA by sequenceanalysis. Methods of DNA sequence analysis are well known in the art. Awell known method of sequencing is the “chain termination” method firstdescribed by Sanger et al., PNAS (USA) 74(12):5463-5467 (1977) anddetailed in SEQUENASE™ 2.0 product literature (Amersham Life Sciences,Cleveland). Sequencing can be performed using a single primer or aprimer pair. Primers are chosen for sequencing based on their proximityto the region of interest. Non-limiting examples of suitable sequencingprimers for each exon are described in Table 2.

Once the nucleic acid sequence from the test subject is obtained, thesequence is compared to the nucleic acid sequence of one or moresubjects not exhibiting peripheral neuropathy in order to identifygenetic mutations in SIMPLE that are associated with peripheralneurological disease. For example, resulting sequences can be alignedwith the known exon sequence using a multiple sequence alignment tool,Sequencher (Gene Codes Corporation, Ann Arbor, Mich.), in order toidentify any nucleotide changes as described in Example 5. In oneembodiment, the information and analysis can be recorded on a databaseand the comparisons can be performed by a computer system accessing saiddatabase. In this manner, the amplified sequences of SIMPLE from asubject exhibiting peripheral neurological disease are sequenced until amutation in SIMPLE associated with peripheral neurological disease isidentified.

A mutation associated with peripheral neurological disease encompassesany alteration of the wild-type small integral membrane protein of thelysosome/late endosome (“SIMPLE”) sequence deposited in GenBank,provided as SEQ ID NO: 3, that is not a recognized polymorphism. Apolymorphism typically has a population frequency of greater than 1% inmammalian control subjects of the same species that do not exhibitperipheral neuropathy, and is not associated with peripheralneurological disease. In contrast, a mutation that is positivelyassociated with peripheral neurological disease typically co-segregateswith family members exhibiting peripheral neuropathy. The followingcharacteristics are supportive, but are not required for a geneticmutation to be a causative mutation for peripheral neurological disease:(1) the change results in an amino acid substitution in a highlyevolutionarily conserved residue of the SIMPLE protein (such as in exon3 (SEQ ID NO: 5) or in the conserved region of exon 3 (SEQ ID NO: 7);(2) the change occurs in a functional domain of SIMPLE; (3) the changeis predicted to affect splicing; or (4) the change co-segregates withdisease in a family in an autosomal dominant manner.

A genetic mutation may be any form of sequence alteration including adeletion, insertion, point mutation or DNA rearrangement in the codingor noncoding regions. Deletions may be small or large and may be of theentire gene or of only a portion of the gene. Point mutations may resultin stop codons, frameshift mutations or amino acid substitutions. Pointmutations may also occur in regulatory regions, such as in the promoterof the SIMPLE gene, leading to loss or diminution of expression of themRNA. Point mutations may also abolish proper RNA processing, leading toloss of expression of the SIMPLE gene product, or to a decrease in mRNAstability or translation efficiency. DNA rearrangements include a simpleinversion of a single segment of DNA, a reciprocal or nonreciprocaltranslocation disrupting any portion of the gene, or a more complexrearrangement.

In one embodiment of this aspect of the method of the invention, once amutation is identified in a subject exhibiting peripheral neuropathy,co-segregation analysis is carried out to determine if the particularmutation in the SIMPLE gene co-segregates with the presence ofperipheral neuropathy in the subjects tested. The standard test forgenetic linkage is described in J. Ott (1999), Analysis of Human GeneticLinkage, 3d ed., The Johns Hopkins University Press. Co-segregationanalysis can be done in several ways. In one embodiment, co-segregationanalysis is done by sequencing DNA amplified from the corresponding exonin subjects exhibiting peripheral neuropathy utilizing the previouslydescribed methods. For example, DNA sequence variations can beidentified using DNA sequencing, as described in Example 1.Alternatively, there are several other methods that can be used todetect and confirm DNA sequence variation including, for example, (1)restriction fragment length polymorphism (RFLP) analysis as described inExample 3; (2) single stranded conformation analysis (SSCA) (Orita etal., Proc. Nat'l. Acad. Sci. USA 86:2776-2770 (1989)); (3) denaturinggradient gel electrophoresis (DGGE) based on the detection of mismatchesbetween the two complementary DNA strands (Wartell et al., Nucl. AcidsRes. 18:2699-2705 (1990)); (4) RNase protection assays (Finkelstein etal., Genomics 7:167-172 (1990)); (5) hybridization with allele-specificoligonucleotides (ASOs) (Conner et al., Proc. Nat'l. Acad. Sci. USA80:278-282 (1983)); and (6) allele-specific PCR (Rano & Kidd, Nucl.Acids Res. 17:8392 (1989)). In the SSCA, DGGE and RNase protectionassay, a new electrophoretic band appears when a mutation is present.SSCA detects a band which migrates differently because the sequencechange causes a difference in single-strand, intramolecular basepairing. DGGE detects differences in migration rates of mutant sequencescompared to wild-type sequences using a denaturing gradient gel. Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular SIMPLE mutation. If the particular SIMPLE mutation isnot present, an amplification product is not observed. Insertions anddeletions of genes can also be detected by cloning, sequencing andamplification.

In another embodiment, genetic mutations are identified by hybridizationof amplified regions of the SIMPLE gene with allele-specificoligonucleotides. For example, a hybridization assay may be carried outby isolating genomic DNA from a mammalian subject exhibiting peripheralneuropathy, contacting the isolated DNA with a hybridization probespecific for a SIMPLE gene mutation under conditions suitable forhybridization of the probe with the isolated genomic DNA, said DNA probespanning said mutation in said gene, wherein said DNA probe is capableof detecting said mutation; and determining the presence or absence ofsaid hybridized DNA probe as an indication of the presence or absence ofsaid genetic mutation. Desirable probes useful in such a DNAhybridization assay comprise a nucleic acid sequence that is unique tothe genetic mutation. Examples of useful DNA probes include SEQ ID NO:6; SEQ ID NO: 8; SEQ ID NO: 10 and SEQ ID NO: 12 as provided in Table 1.Analysis can involve denaturing gradient gel electrophoresis ordenaturing HPLC methods, for example. For guidance regarding probedesign and denaturing gel electrophoresis or denaturing HPLC methods,see, e.g., Ausubel et al., 1989, Current Protocols in Molecular Biology,Green Publishing Associates and Wiley Interscience, N.Y.

In another embodiment of this aspect of the method of the invention,restriction fragment length polymorphism (RFLP) for the SIMPLE gene canbe used to score for a genetic mutation in a co-segregation analysis.RFLP has been described in U.S. Pat. Nos. 4,965,188 and 4,800,159,incorporated herein by reference. In this technique, restriction enzymesare used which provide a characteristic pattern of restrictionfragments, wherein a restriction site is either missing or an additionalrestriction site is introduced in the mutant allele. Thus, DNA from anindividual and from control DNA sequences are isolated and subjected tocleavage by restriction enzymes which are known to provide restrictionfragments which differentiate between normal and mutant alleles, and therestriction patterns are identified. Example 3 and Table 4 furtherillustrate RFLP methods that are useful in the practice of the method ofthe invention.

Several genetic mutations in SIMPLE that are associated with theperipheral neurological disease CMT1 have been identified by practicingthe methods of this aspect of the invention as described in Examples 1and 2 and shown in Tables 1 and 3. Table 3 provides a list of mutationsidentified in a SIMPLE gene using the methods of this aspect of theinvention. The first column of Table 3 describes the exon each mutationresides in, the second column describes the nucleotide change in thecDNA (numbered sequentially with reference to SEQ ID NO: 1) for eachmutant, the third column describes the type of mutation that is present,the fourth column describes primer pairs useful to PCR amplify the exoncontaining the mutation, and the fifth column describes primers usefulfor sequencing across the region containing the mutation.

TABLE 3 SUMMARY OF MUTATIONS IDENTIFIED IN THE SIMPLE GENE THATCO-SEGREGATE WITH PERIPHERAL NEUROPATHY Predicted Nucleotide amino acidchange in change in Type of Primers used Primers used to Exon cDNAprotein Mutation to PCR amplify sequence 3 334G to A G112S missense 3F(SEQ ID NO: 18) 3F (SEQ ID NO: 18) 3R (SEQ ID NO: 19) 3 344C to A T115Nmissense 3F (SEQ ID NO: 18) 3F (SEQ ID NO: 18) 3R (SEQ ID NO: 19) 3 346Tto G W116G missense 3F (SEQ ID NO: 18) 3F (SEQ ID NO: 18) 3R (SEQ ID NO:19)

In another aspect, the present invention provides isolated nucleic acidmolecules encoding a SIMPLE protein comprising a mutation selected fromthe group consisting of G112S, T115N, and W116G. The mutations in theSIMPLE protein are numbered sequentially according to the first aminoacid of SEQ ID NO: 2. The nucleotide sequences are numbered sequentiallyaccording to the first nucleotide of SEQ ID NO: 1. Each mutation isfurther described as follows:

Mutation G112S results from a nucleotide change of G to A at nucleotide334, which results in a codon change from GGT to AGT which in turnresults in the missense mutation at amino acid G112 to S, substituting aserine for a glycine at amino acid position 112 in the SIMPLE protein.In some embodiments, mutation G112S is encoded by SEQ ID NO: 8 as shownin Table 1.

Mutation T115N results from a nucleotide change of C to A at nucleotide344, which results in a codon change from ACC to AAC which in turnresults in the missense mutation at amino acid T115 to N, substitutingan asparagine for a threonine at amino acid position 115 in the SIMPLEprotein. In some embodiments, mutation T115N is encoded by SEQ ID NO: 10as shown in Table 1.

Mutation W116G results from a nucleotide change of T to G at nucleotide346, which results in a codon change from TGG to GGG which in turnresults in the missense mutation at amino acid W116 to G, substituting aglycine for a tryptophan at amino acid position 116 in the SIMPLEprotein. In some embodiments, mutation W116G is encoded by SEQ ID NO: 12as shown in Table 1.

In some embodiments, the isolated nucleic acid molecules describedherein comprise a sequence selected from the group consisting of SEQ IDNO: 8, SEQ ID NO: 10, and SEQ ID NO: 12. In this regard, in someembodiments, the isolated nucleic acid molecules described herein are atleast 90% identical to a portion of SEQ ID NO: 1 or its complement. Insome embodiments, the isolated nucleic acid molecules described hereinare at least 90% identical to a portion of SEQ ID NO: 3 or itscomplement. In some embodiments, the isolated nucleic acid moleculesdescribed herein are at least 90% identical to a portion of SEQ ID NO: 4or its complement. In some embodiments, the isolated nucleic acidmolecules described herein are at least 90% identical to an isolatednucleic acid molecule selected from the group consisting of SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12 as described inTable 1. In some embodiments, the isolated nucleic acid moleculesdescribed herein hybridize to the complement of SEQ ID NO: 1 underconditions of 5×SSC at 50° C. for 1 hr. In some embodiments, theisolated nucleic acid molecules described herein hybridize to thecomplement of SEQ ID NO: 1 under conditions of 5×SSC at 60° C. for 1 hr.In some embodiments, the isolated nucleic acid molecules describedherein hybridize to the complement of SEQ ID NO: 3 under conditions of5×SSC at 50° C. for 1 hr. In some embodiments, the isolated nucleic acidmolecules described herein hybridize to the complement of SEQ ID NO: 3under conditions of 5×SSC at 60° C. for 1 hr. In some embodiments, theisolated nucleic acid molecules described herein hybridize to thecomplement of SEQ ID NO: 4 under conditions of 5×SSC at 50° C. for 1 hr.In some embodiments, the isolated nucleic acid molecules describedherein hybridize to the complement of SEQ ID NO: 4 under conditions of5×SSC at 60° C. for 1 hr.

Some nucleic acid embodiments, for example, include genomic DNA, RNA,and cDNA encoding the mutant proteins or fragments thereof. In someembodiments, the invention also encompasses DNA vectors such as, forexample DNA expression vectors that contain any of the foregoing nucleicacid sequences operatively associated with a regulatory element thatdirects the expression of the coding sequences the nucleic acids above,and genetically engineered host cells that contain any of the foregoingnucleic acid sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences in the host cell.The nucleic acids encoding the SIMPLE protein mutations can bemanipulated using conventional techniques in molecular biology so as tocreate recombinant constructs that express mutant polypeptides.

The nucleic acid sequences described above have diagnostic as well astherapeutic use. The nucleic acid sequences can be used as probes toidentify more genetic mutations in the SIMPLE gene and to detect thepresence or absence of wild-type or mutant genes in an individual, suchas in nucleic acid hybridization assays, southern and northern blotanalysis, and as controls for screening assays and the kits describedherein. The sequences described herein can also be incorporated intoconstructs for preparing recombinant mutant proteins or used in methodsof searching or identifying agents that modulate SIMPLE levels and/oractivity, for example, candidate therapeutic agents. For example, agentsthat modulate SIMPLE levels may be utilized to treat diseases of thenervous system. Because the mutations of this aspect of the inventionare dominant negative or gain of function mutations, they have also havetherapeutic utility for use in the identification and development anddesign of drugs which circumvent or overcome the mutated SIMPLE genefunction. The sequences of the nucleic acids and/or proteins describedherein can also be incorporated into computer systems and used withmodeling software so as to enable rational drug design. Information fromgenotyping methods provided herein can be used, for example, in computersystems, in pharmacogenomic profiling of therapeutic agents to predicteffectiveness of an agent in treating an individual for a neurologicaldisease.

The identification of mutants T115N and G112S is described in Example 1.The identification of mutant W116G is described in Example 2. Theco-segregation analysis of these mutations is described in Example 3 andTable 4. Further characterization of these mutations is described inTables 1 and 3.

In another aspect, the invention provides a nucleic acid probe fordetecting a SIMPLE gene, the probe consisting of a nucleic acid sequenceselected from the group consisting of nucleotide 91 to nucleotide 140 ofSEQ ID NO: 4, or the complement thereof and SEQ ID NO: 6, or thecomplement thereof. The nucleic acid probes of this aspect of theinvention are useful to detect the presence of a wild-type gene in anindividual, such as in nucleic acid hybridization assays, southern andnorthern blot analysis and as controls for the screening assays and kitsdescribed herein. The nucleic acid probes of this aspect of theinvention may be used in nucleic acid hybridization assays with genomicDNA isolated from a mammalian subject as described herein.

In another aspect, the invention provides nucleic acid primer moleculesconsisting of sequence SEQ ID NO: 18 and SEQ ID NO: 19. The primermolecules of the invention can be used individually as sequencingprimers, or together as a primer pair for amplifying exon 3 of theSIMPLE gene, under conditions as disclosed in Table 2. SEQ ID NO: 18 andSEQ ID NO: 19 can be used, for example, to screen for mutations in theSIMPLE gene that are associated with peripheral neuropathy and areuseful reagents in the methods and kits described herein.

In another aspect, the invention provides isolated mutant SIMPLEpolypeptides and peptide fragments. Mutant SIMPLE polypeptides areSIMPLE proteins encoded by a SIMPLE gene having at least one of themutations associated with peripheral neuropathy, as described above. Insome embodiments, the isolated polypeptide includes mutation G112S, suchas, for example, an isolated polypeptide comprising SEQ ID NO: 9. Inother embodiments, the isolated polypeptide includes mutation T115N,such as, for example, an isolated polypeptide comprising SEQ ID NO: 11.In further embodiments, the isolated polypeptide includes mutationW116G, such as, for example, an isolated polypeptide comprising SEQ IDNO: 13. The isolated mutant SIMPLE polypeptides and peptide fragmentsare useful, for example, as antigens for raising antibodies whichspecifically bind to mutant SIMPLE polypeptides.

In another aspect, the present invention provides methods of screening amammalian subject to determine if said subject has a geneticpredisposition to develop, or is suffering from Charcot-Marie-Tooth type1C (CMT1C) neuropathy. The methods of this aspect of the inventioncomprise the step of analyzing the nucleic acid sequence of a SIMPLEgene in a subject to determine whether a genetic mutation that isassociated with CMT1C is present in the nucleic acid sequence, whereinthe presence of such a mutation indicates that the mammalian subject hasa genetic predisposition to develop CMT1C or is diagnosed as sufferingfrom such a disease. In some embodiments, the method further comprisesdetermining whether the mammalian subject is exhibiting peripheralneuropathy. The clinical examination of a mammalian subject for symptomsrelated to peripheral neuropathy may be done either prior to, or afternucleic acid analysis of the test subject.

The method of this aspect of the invention is useful for screening anymammalian subject, such as for example, a human subject, for the geneticpredisposition to develop CMT1C disease. The method is useful forpreimplantation, prenatal and postnatal diagnosis of neurologicaldisease caused by mutation in the SIMPLE gene that facilitates geneticcounseling and therapeutic intervention. The method is especially usefulfor screening and diagnosing presymptomatic at-risk family members forthe presence or absence of mutations in SIMPLE associated with thedisease. The method is also useful for screening subjects exhibitingperipheral neuropathy to determine whether their symptoms are caused bya genetic mutation in the SIMPLE gene.

Any genetic mutation in the SIMPLE gene that co-segregates with CMT1C isuseful in the practice of the method of this aspect of the invention.Examples of such mutations are shown in Table 3. In one embodiment,genetic mutations that co-segregate with CMT1C are missense mutations inwhich a nucleic acid base change results in an amino acid substitutionin the SIMPLE protein. Examples of such missense mutations include, forexample, G112S, T115N, and W116G as shown in Table 3.

In another embodiment, the method of this aspect of the invention can bepracticed using mutations that cause deletions or silent mutations whichdo not alter the amino acid sequence, but may change splicing or generegulation.

In some embodiments of the invention, subjects are screened for geneticmutations at one or more of the SIMPLE protein positions: 111, 112, 113,114, 115, or 116.

In some embodiments of this aspect of the method of the invention,subjects are screened for the presence of a genetic mutation that isassociated with CMT1C disease in exon 3 of a SIMPLE gene, such as, forexample, nucleotides 32,685 to 32,840 of SEQ ID NO: 3. Exon 3 encodes aregion of highly conserved amino acid residues as shown in Table 1,provided as SEQ ID NO: 7. Examples of mutations found in exon 3 withinthe highly conserved region of SEQ ID NO: 7 that co-segregate with CMT1Care shown in Table 1 and include G112S, T115N, and W116G.

Individuals carrying particular mutations in the SIMPLE gene may beidentified using a variety of techniques of analyzing nucleic acidsequence that are well known in the art such as, for example, directsequencing, PCR amplification and sequencing, restriction fragmentlength polymorphism (RFLP), nucleic acid hybridization, and singlestrand conformation polymorphism (SSCP). For each of these techniques,the test subject provides a biological sample containing genomic DNA tobe analyzed. The test sample may be obtained from body cells, such asthose present in peripheral blood, cheek cells, urine, saliva, surgicalspecimen, and autopsy specimens. The test sample can be processed toinactivate interfering compounds, and to purify or partially purify thenucleic acids in the sample. Any suitable purification method can beemployed to obtain purified or partially purified nucleic acids from thetest sample. A lysing reagent optionally can be added to the sample,particularly when the nucleic acids in the sample are sequestered orenveloped, for example, by cellular or nuclear membranes. Additionally,any combination of additives, such as buffering reagents, suitableproteases, protease inhibitors, nucleases, nuclease inhibitors anddetergents can be added to the sample to improve the amplificationand/or detection of the nucleic acids in the sample. Additionally, whenthe nucleic acids in the sample are purified or partially purified, theuse of precipitation can be used, or solid support binding reagents canbe added to or contacted to the sample, or other methods and/or reagentscan be used. One of ordinary skill in the art can routinely select anduse additives for, and methods for preparation of a nucleic acid samplefor amplification.

In one embodiment of the method of the invention, the nucleic acidsequence is analyzed by direct sequencing for differences in nucleicacid sequence from the wild-type SIMPLE gene by sequencing of thesubject's SIMPLE gene using primers specific for the region of interest,such as, for example, the sequencing primers described in Table 2 andTable 3.

In another embodiment, prior to sequencing the DNA is amplifiedenzymatically in vitro through use of PCR (Saiki et al., Science239:487-491 (1988)) or other in vitro amplification methods aspreviously described herein. In a further embodiment, the DNA from anindividual can be evaluated using RFLP techniques are described inExample 3 and elsewhere herein. The previously described methods usefulfor determining co-segregation analysis are also useful in this aspectof the method of the invention, such as, for example, nucleic acidhybridization techniques and single strand conformation polymorphism(SSCP). SSCP is a rapid and sensitive assay for nucleotide alterations,including point mutations (see Orita, M., et al., Genomics 5:874-879(1989)). DNA segments ranging in length from approximately 100 bp toapproximately 400 bp are amplified by PCR, heat denatured andelectrophoresed on high resolution-non-denaturing gels. Under theseconditions, each single-stranded DNA fragment assumes a secondarystructure determined in part by its nucleotide sequence. Even singlebase changes can significantly affect the electrophoretic mobility ofthe PCR product.

In another aspect, the present invention provides kits for determiningsusceptibility or presence of CMT1C in a subject. The kits of theinvention include (i) one or more nucleic acid primer molecules foramplification of a portion of the SIMPLE gene; and (ii) written indiciaindicating a correlation between the presence of said mutation and riskof developing CMT1C. In one embodiment, the kits of the inventionfurther comprise means for determining whether a mutation associatedwith CMT1C is present. In some embodiments, the kits of the inventioncomprise detection components specific for one or more of the particulargenetic mutations described herein.

Primer molecules for amplification of a portion of the SIMPLE gene canbe of any suitable length and composition and are selected to facilitateamplification of at least one or more regions (in the case of duplexedor multiplexed amplification) of the SIMPLE gene as shown in SEQ ID NO:3 that potentially contains a genetic mutation. For example,oligonucleotide primers can be in the range of 5 bp to 50 bp or longer,and are chosen as primer pairs so that primers hybridize to sequencesflanking the putative mutation. Primer pairs typically have an annealingtemperature within about 20° C. of each other. Computer programs areuseful in the design of primers with the required specificity andoptimal amplification properties. See, e.g., Oligo version 5.0(available from National Biosciences Inc., 3001 Harbor Lane, Suite 156,Plymouth, Minn.). Examples of primer pairs suitable for inclusion in thekit of the invention are provided in Table 2.

Similarly, a kit of the invention can also provide reagents for aduplexed amplification reaction (with two pairs of primers) amultiplexed amplification reaction (with three or more pair of primers)so as to amplify multiple sites of SIMPLE nucleotide mutations in onereaction.

Also included in the kit of the invention are written indicia indicatinga correlation (typically a positive correlation) between the presence ofa particular mutation in the SIMPLE gene and the risk of developingCMT1C disease.

The kit optionally also comprises one or more enzymes useful in theamplification or detection of nucleic acids and/or nucleotide sequences.Suitable enzymes include DNA polymerases, RNA polymerases, ligases, andphage replicases. Additional suitable enzymes include kinases,phosphatases, endonucleases, exonucleases, RNAses specific forparticular forms of nucleic acids (including, but not limited to, RNAseH), and ribozymes. Other suitable enzymes can also be included in thekit.

The kit optionally comprises amplification reaction reagents suitablefor use in nucleic acid amplification. Such reagents are well known andinclude, but are not limited to: enzyme cofactors such as magnesium ormanganese; salts; nicotinamide adenine dinucleotide (NAD), anddeoxynucleoside triphosphates (dNTPs). The kit optionally can alsocomprise detection reaction reagents, such as light or fluorescencegenerating substrates for enzymes linked to probes.

The kit optionally includes control DNA, such as positive and negativecontrol samples. Negative control samples may comprise for example,genomic DNA or SIMPLE cDNA from a mammalian subject with nopredisposition to CMT1C, or portions thereof. Positive control samplesmay comprise, for example, nucleic acid molecules containing anidentified mutation in the SIMPLE gene as described herein.

The kit optionally includes instructions for using the kit in thedetection of mutations in SIMPLE associated with CMT1C disease. The kitalso preferably includes instructions on the appropriate parameters forthe amplification reaction. Any suitable set of amplification parameterscan be employed. For example, the precise temperature at whichdouble-stranded nucleic acid sequences dissociate, primers hybridize ordissociate, and polymerase is active, are dependent on the length andcomposition of the sequences involved, the salt content of the reaction,the oligonucleotide concentration, the viscosity of the reaction and thetype of polymerase. One of ordinary skill in the art can easilydetermine appropriate temperatures for the amplification reaction (see,e.g., Wetmur, J. Critical Reviews in Biochemistry and Molecular Biology26:227-59 (1991)). For example, temperatures above about 90° C., such asbetween about 92° C., and about 100° C., are typically suitable for thedissociation of double-stranded nucleic acid sequences. Temperatures forforming primer hybrids are preferably between about 45° C. and about 65°C. Temperatures for the polymerization/extension phase are typicallybetween about 60° C. and about 90° C., depending on the polymeraseutilized in the reaction.

A multiplicity of suitable methods may be used to analyze the amplifiednucleic acid product to determine whether a mutation associated withCMT1C disease is present. Suitable means include DNA sequencing,northern blotting, southern blotting, Southwestern blotting, probe shiftassays (see, e.g., Kumar et al., AIDS Res. Hum. Retroviruses 5:345-54(1989), T4 Endonuclease VII-mediated mismatch-cleavage detection (see,e.g., Youil et al., Proc. Nat'l. Acad. Sci. USA 92:87-91 (1995),Fluorescence Polarization Extension (FPE), Single Strand LengthPolymorphism (SSLP), PCR-Restriction Fragment Length Polymorphism(PCR-RFLP), Immobilized Mismatch Binding Protein Mediated(MutS-mediated) Mismatch detection (see, e.g., Wagner et al., NucleicAcids Research 23:3944-48 (1995), reverse dot blotting, (see, e.g.,European Patent Application No. 0 511 559), hybridization-mediatedenzyme recognition (see, e.g., Kwiatkowski et al., Mol. Diagn.4(4):353-64 (1999)), describing the Invader™ embodiment of thistechnology by Third-Wave Technologies, Inc.), detection, single-strandconformation polymorphism (SSCP) and gradient denaturing gelelectrophoresis to detect probe-target mismatches (e.g., “DGGE”, see,e.g., Abrams et al., Genomics 7:463-75 (1990), Ganguly et al., Proc.Nat'l. Acad. Sci. USA 90:10325-29 (1993), and Myers et al., MethodsEnzymology 155:501-27 (1987)).

The kit is preferably provided in a microbiologically stable form.Microbiological stability can be achieved by any suitable means, such asby (i) freezing, refrigeration, or lyophilization of kit components;(ii) by heat-, chemical-, or filtration-mediated sterilization orpartial sterilization; and/or (iii) by the addition of antimicrobialagents such as azide, detergents, and other suitable reagents to otherkit components. The kit can also be optionally provided in a suitablehousing that is preferably useful for robotic handling by a clinicallyuseful sample analyzer. For example, the kit can optionally comprisemultiple liquids, each of which are stored in distinct compartmentswithin the housing. In turn, each compartment can be sealed by a devicethat can be removed, or easily penetrated, by a mechanical device.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention. All literature citations herein are expressly incorporated byreference

Example 1

This example describes the identification of the T115N and the G112Smissense mutations in the SIMPLE gene and demonstrates that thesemutations co-segregate with Charcot-Marie-Tooth neuropathy type 1C.

Mapping an Autosomal Dominant Charcot-Marie-Tooth Neuropathy toChromosome 16p

Subjects:

A four generation family of Irish descent comprising 37 family members,some of which exhibited unexplained Charcot-Marie-Tooth neuropathy wasidentified and designated pedigree K1550 (see Street et al., Am J. Hum.Genet. 70:244-250 (2002)). Another four generation family of Englishdescent with 38 family members also comprising some family membersexhibiting unexplained Charcot-Marie-Tooth neuropathy was identified anddesignated pedigree K1551. Id. Affected family members met widelyaccepted criteria for CMT1 disease including distal muscle weakness andatrophy, depressed deep tendon reflexes and sensory impairment (see Dyckand Lambert, Arch. Neurol. 18:603-618 (1968)). The mean ulnar (16.7 m/s[n=3], 25.3 [n=8]), median (23 m/s [n=5], 25.8 m/s [n=12]) and peroneal(20.4 m/s [n=4, 21 m/s [n=6]) motor nerve conduction velocities ofaffected K1550 and K1551 patients were consistent with CMT1 (see Streetet al., Neurology 60:22-26 (2003)). One affected individual in pedigreeK1550 had a sural nerve biopsy taken during reconstructive foot surgerythat demonstrated “onion-bulb hypertrophy” typical of demyelinating CMT.Id. 200 unrelated control DNA samples for mutational analysis were takenfrom a collection of predominantly Caucasians of European descent.

Mapping and Identification of SIMPLE as a Candidate for CMT1:

To identify the locus responsible for the phenotype in these families, awhole genome-wide scan was performed in pedigrees K1550 and K1551, withinformative microsatellite markers spaced at 10 cM intervals using themethods as described in Street et al., Am. J. Med. Genet. 70:244-250(2002). Using two markers, D165764 and D165519 (obtained from ResearchGenetics), the CMT1 gene was mapped to chromosome 16p within a 9-cMinterval. Id. SIMPLE was identified as one of 20 candidate genes thatmapped to the critical region on chromosome 16p and was evaluated forDNA sequence alterations in families K1550 and K1551 (Street et al.,Neurology 60:22-26 (2003)).

Molecular Analysis:

The following protocol of informed consent was approved by theinstitutional review board (IRB) of the University of Washington,Seattle. 15 to 20 mL of blood was obtained by venipuncture forhigh-molecular weight DNA (as described by Neitzel et al., Hum. Genet.73:320-326 (1986)) and used as a template for PCR amplification. Thethree coding exons of the SIMPLE gene were PCR amplified from subjectgenomic DNA utilizing primer pairs listed in Table 2. PCR reactions werecarried out in 25 μl containing 1×PCR buffer of 10 mM Tris-HCL (pH 8.3at 25° C.), 50 mM KCL, 2 mM MgCl₂, 0.2 mM each dNTP (dATP, dCTP, dGTP,dTTP), 0.66 μM each oligonucleotide forward and reverse primer, and 0.6U of 5 U/μl Ampli-Taq Polymerase (Sigma, St. Louis, Mo.). 5 μl of PCRproduct was characterized by gel electrophoresis/ethidium bromidestaining for the presence of a single correctly sized band, as shown inTable 2.

Direct DNA Sequencing of the PCR Fragments:

5 μl of PCR product from each sample confirmed to have a singlecorrectly sized band was treated with 1 μl of ExoSAP-IT (US Biochemical,Cleveland, Ohio) at 37° C. for 2 hours followed by heat inactivation at85° C. for 10 minutes. Direct DNA sequencing of the purified fragmentswas carried out by using a BigDye Terminator Cycle Sequencing ReadyReaction Kit (Applied Biosystems Inc., Foster City, Calif.). The primersused for sequencing are the same primers used for PCR amplification aslisted in Table 2. For initial mutation screening, either forward orreverse primer was used. The PCR reaction contained 3 μl of treated PCRproduct (˜100 ng), 3 pmol primer, 1 μl sequencing buffer and 2 μl ofBigDye reagent in a total volume of 10 μl. The sequencing reaction wascarried out in a PTC-100 Programmable Thermal Controller (MJ ResearchInc., Waltham, Mass.) with cycle conditions of 96° C. for 2 min, 30cycles of 96° C. for 15 sec, 50° C. for 10 sec, and 60° C. for 4 min.The sequencing product was purified by ethanol/EDTA precipitation, thenelectrophoresed on an ABI DNA Sequencer (Applied Biosystems Inc., FosterCity, Calif.).

Results:

Analysis of the three SIMPLE coding exons and flanking intron nucleotidesequences in pedigrees K1550 and K1551 revealed mutations in exon 3. InK1550, a C to A transversion at nucleotide 344 (as counted from the cDNAstart codon as shown in SEQ ID NO: 1) was detected in exon 3, whichpredicts substitution of asparagine for threonine at amino acid position115 (T115N).

In K1551, a G to A transition at nucleotide 344 was detected in exon 3,which predicts substitution of serine for glycine at amino acid position112 (G112S).

Evaluation of Co-Segregation of CMT1C and Genetic Mutations

The C to A transversion at nucleotide 344 and the G to A transition atnucleotide 334 each introduced a novel BsrI restriction endonucleasesite which was utilized to verify that the mutation co-segregates withsubjects exhibiting peripheral neuropathy as further described inExample 3 and Table 4.

Example 2

This example describes the identification of the W116G missense mutationin the SIMPLE gene.

Subjects Tested:

A family of Dutch descent comprising 17 family members, some of whichexhibited unexplained Charcot-Marie-Tooth neuropathy was identified anddesignated pedigree K2900 (see Street et al., Neurology 60:22-26(2003)). The proband in this family had decreased nerve conductionvelocities ranging from 15 to 30 m/s, consistent with CMT1C. Affectedfamily members had previously been evaluated and shown not to havealterations in the PMP22, MPZ or EGR2 genes. 100 unrelated controlchromosomes were also included in the study.

Methods:

The entire coding region of SIMPLE was sequenced in genomic DNA of oneaffected individual by first PCR amplifying the 3 coding exons (exons2-4) and sequencing each using the primers shown in Table 2 as describedin Example 1.

Results:

In K2900, a T to G transversion at nucleotide 346 (as counted from thecDNA start codon as shown in SEQ ID NO: 1) was detected in exon 3, whichpredicts substitution of glycine for tryptophan at amino acid position116 (W116G).

Evaluation of Co-Segregation of CMT1C and Genetic Mutations

The T to G transversion at nucleotide 346 introduced a novel Nci1restriction endonuclease site which was utilized to verify that themutation co-segregates with subjects exhibiting peripheral neuropathy asfurther described in Example 3 and Table 4.

Example 3

This example describes the use of restriction fragment lengthpolymorphism (RFLP) analysis to evaluate co-segregation of peripheralneuropathy with genetic mutation in the SIMPLE gene.

Restriction Fragment Length Polymorphism (RFLP) Analysis:

Each of the identified mutations, G112S, T115N and W116G, alter therestriction endonuclease digestion pattern of specific restrictionendonucleases as shown in Table 4. The first column of Table 4 describesthe mutations amenable to RFLP analysis, the second column provides auseful primer set for amplification of the region encompassing themutation, the third column provides the relevant restrictionendonuclease for use in digestion of the PCR fragment, and the fourthand fifth columns provide the expected restriction enzyme digestedfragments for wild-type and mutant genes, respectively. The final twocolumns of Table 4 provide the reaction conditions appropriate for eachrestriction enzyme digestion listed.

Results of Segregation Analysis:

G112S Mutation:

RFLP analysis was performed on samples from 33 individuals from theK1551 pedigree (described in Example 1), including 18 individualsexhibiting demyelinating peripheral neuropathy. Bsr1 digestion of the380 bp exon 3 fragment resulted in the pattern shown in Table 4 formutant-type samples in all 18 subjects exhibiting peripheral neuropathy.Of the 15 samples from individuals not exhibiting peripheral neuropathy,all Bsr1 restriction patterns corresponded to the expected fragmentpattern for wild-type shown in Table 4. The expected wild-type fragmentpattern was observed in 200 unrelated samples of control chromosomes.

T115N Mutation:

RFLP analysis was performed on samples from 29 individuals from theK1550 pedigree (described in Example 1), including 21 individualsexhibiting peripheral neuropathy. Bsr1 digestion of the 380 bp exon 3fragment resulted in the pattern shown in Table 4 for mutant-typesamples in all 21 subjects exhibiting peripheral neuropathy. Of the 8samples from individuals not exhibiting peripheral neuropathy, all Bsr1restriction patterns corresponded to the expected fragment pattern forwild-type shown in Table 4. The expected wild-type fragment pattern wasobserved in 200 unrelated samples of control chromosomes.

W116G Mutation:

RFLP analysis was performed on samples from 8 individuals from the K2900pedigree (described in Example 2), including 4 individuals exhibitingperipheral neuropathy. Nci1 digestion of the 380 bp exon fragmentresulted in the pattern shown in Table 4 for mutant-type samples in all4 individual subjects exhibiting peripheral neuropathy. Of the 4 samplesfrom individuals not exhibiting peripheral neuropathy, all Nci1restriction patterns corresponded to the expected fragment pattern forwild-type shown in Table 4. The expected wild-type fragment pattern wasobserved in 100 unrelated samples of control chromosomes.

TABLE 4 THE CONDITIONS OF RFLP ANALYSIS FOR DETERMINING COSEGREGATIONWITH DEMYELINATING NEUROPATHY Restriction Fragment Sizes (bp) ConditionsMutation Primer Set Enzyme Wild-type Mutant Temp Buffer 334G to A 3F(SEQ ID NO: 18) Bsr1 380 121, 259 65° C. NEBuffer 3 (G112S) 3R (SEQ IDNO: 19) 344C to A 3F (SEQ ID NO: 18) Bsr1 380 108, 272 65° C. NEBuffer 3(T115N) 3R (SEQ ID NO: 19) 346T to G 3F (SEQ ID NO: 18) Nci1 380 104,276 37° C. NEBuffer 4 (W116G) 3R (SEQ ID NO: 19)

Example 4

This example describes the analysis of SIMPLE gene expression afternerve injury in a rat model.

Methods:

Young adult Sprague-Dawley rats were anesthetized and the sciatic nerveswere transected at the sciatic notch, and both cut ends were ligated andpulled apart to prevent axonal regeneration into the distal stump. Theentire distal nerve stump (about 4 cm in length) was harvested duringthe next 1 to 58 days later and divided into 2-cm segments, termed P(the segment immediately adjacent to the crush) and D (the more distalsegment). Further description of this experimental method is provided inScarlato et al., J Neurosci. Res. 66:16-22 (2001).

RNA Expression Analysis:

RNA Isolation:

RNA was isolated by CsCl₂ gradient centrifugation as described byChirgwin et al., Biochem. 18:5294-5299 (1979). For the lesioned adultrat sciatic nerves, total RNA was isolated from distal stumps of sciaticnerves that were transected or crushed and a Northern Blot was probedwith the following cDNAs: a 1.4 kb fragment of SIMPLE, a full-lengthcDNA of rat myelin protein zero; and a full-length cDNA of rat GAPDH.

Results:

Northern blot analysis indicated that the 2.4 kb SIMPLE message waspresent at moderate levels in rat sciatic nerve, with expressionremaining constant during sciatic nerve development. Following axotomyof transected sciatic nerve, SIMPLE expression remained essentiallyconstant for a 48 day time course. Following crush injury, a generalincrease in SIMPLE expression was observed over a 58 day time course,and was more pronounced in the nerve region proximal to the site ofinjury. The fact that SIMPLE expression was unchanged as a result ofnerve injury stands in distinct contrast to other CMT1 genes such asMPZ, PMP22, connexin-32 and EGR2, all of which have been found todemonstrate altered expression as a result of nerve injury (see Shereret al., J. Neurosci. 15:8281-8294 (1995); Snipes et al., J. Cell. Biol.117:225-238 (1992) and Zorick et al., Mol. Cell. Neurosci. 8:129-145(1996)).

Protein Expression:

Blood samples were cleared of red blood cells by lysis in osmotic buffer(PureGene). Intact lymphocytes remaining in the lysate were thenpelleted by centrifugation, washed in phosphate-buffered saline, andlysed in boiling SDS-PAGE loading buffer. 50 μg of each extract wasresolved on a SDS-PAGE gel and transferred to PVDF membrane. Blots werethen incubated with anti-LITAF monoclonal antibodies (Transduction Labs;1:5000), followed by horseradish peroxidase-conjugated goat anti-mouseantibodies (Sigma; 1:20,000). Detection was performed with the ECL Plussystem (Amersham).

Results:

Western blot analysis of peripheral blood lymphocytes indicated that theT115N and W116G substitutions do not appear to alter the SIMPLE proteinlevel compared to a control individual and an individual carrying thePMP22 duplication. This result is in contrast to the observation thatoverexpression of the PMP22 gene in CMT1A is associated withdemyelination and formation of perinuclear protein aggregates (Matsumamiet al., Nat. Genet. 1:176-179 (1992)).

Example 5

This example describes a kit and method of use for identifying geneticmutations associated with peripheral neurological disease in a mammaliansubject, and for determining susceptibility or presence of CMT1C in atest subject.

Methods Utilized:

PCR Amplification:

Carried out as described in Example 1

Direct Sequencing:

Carried out as described in Example 1

Data Analysis:

The resulting sequences are aligned with the known exon sequence using amultiple sequence alignment tool, Sequencher (Gene Codes Corporation,Ann Arbor, Mich.), in order to identify any nucleotide changes.Electrophergrams are also visually examined to detect heterozygous basechanges that might be missed by Sequencher.

Confirmation of the Nucleotide Changes:

Once a nucleotide change is detected, the exon fragment encompassing thesuspected mutation is subjected to PCR amplification and directsequencing again, using both forward and reverse primers as shown inTable 2.

For familial cases, when the nucleotide change is confirmed, withconsent, the available family members, including affected and at riskunaffected individuals, are tested to confirm that the mutationsegregates with the disease. After appropriate consent for clinicaltesting is obtained, the test may also be used for presymptomaticdiagnosis in at-risk individuals.

Contents of the SIMPLE Mutation Kit:

-   -   1. 10×PCR Buffer (100 mM Tris-HCL (pH 8.3 at 25° C.), 500 mM        KCL, 20 mM MgCl2    -   2. dNTP mix: dATP, dCTP, dGTP, dTTP at 10 mM each (Sigma, St.        Louis, Mo.)    -   3. Ampli-Taq DNA Polymerase (Sigma, St. Louis, Mo.)    -   4. Primers for amplification of each SIMPLE exon and the        adjacent intronic sequences at 0.66 μM each (as shown in Table        2)    -   5. Exo-SAP-IT (US Biochemical, Cleveland, Ohio)    -   6. BigDye Terminator Cycle Sequencing Ready Reaction Kit        (Applied Biosystems Inc., Foster City, Calif.)    -   7. Control DNA    -   8. Written instructions and indicia indicating a positive        correlation between the presence of a particular mutation in the        SIMPLE gene and the risk of CMT1C disease.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of identifyinga genetic mutation in a human small integral membrane protein of thelysosome/late endosome (“SIMPLE”) gene in a human subject, said methodcomprising: (a) hybridizing a nucleic acid sequence of a coding regionof a human small integral membrane protein of the lysosome/late endosome(“SIMPLE”) gene consisting of a coding portion of SEQ ID NO:1, or anaturally occurring sequence alteration thereof from a human subjectwith an oligonucleotide specific for a SIMPLE gene missense mutation,wherein the missense mutation alters the encoded amino acid sequence ata residue selected from the group consisting of amino acid residues 112,115, and 116 of SEQ ID NO:2; and (b) detecting hybridization of theoligonucleotide with the nucleic acid sequence from the human subject;and (c) identifying a genetic mutation in the SIMPLE gene in the subjectwhen hybridization is detected in step (b).
 2. The method of claim 1,wherein the missense mutation is a dominant mutation.
 3. The method ofclaim 1, wherein the missense mutation encodes an altered SIMPLE aminoacid sequence at residue 112, resulting in G112S.
 4. The method of claim1, wherein the missense mutation an altered SIMPLE amino acid sequenceat residue 115, resulting in T115N.
 5. The method of claim 1, whereinthe missense mutation encodes an altered SIMPLE amino acid sequence atresidue 116, resulting in W116G.